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20 Pharmaceutical Technology ® Bio/Pharma Outsourcing Innovation eBook February 2024 PharmTech.com Biologics drove this innovation, as well as the availability of these self-cleaving sequences. The future of affinity ligands PharmTech: In what direction is affinity ligand devel- opment headed moving forward? Shamlou (Abzena): Another popular affinity ligand for purification of antibodies is Protein G, which has a molecular mass of 21.9 kDa and has a different binding affinity to antibodies compared to Protein A. Affinity ligand is a powerful technology and, beyond Protein A and G affinity ligands, in recent years, af- finity chromatography matrices have been developed with high selectivity for purification of, for example, recombinant viral vectors for gene therapy applica- tions. In one example, camelid single domain anti- bodies that recognize adeno-associated virus (AAV) viral vector capsids are proving very effective. Another affinity matrix also provides high affinity for recom- binant human growth hormone (hHGH) by using an immobilized 14 kD single variable domain of heavy chain (VHH) antibody that is recombinantly produced in yeast. The single-domain heavy chain antibody frag- ments from Camelidae have also been utilized success- fully as affinity ligands for purification coagulation factors to help patients with hemophilia type A and B. Elliott (BioCina): Several factors must be considered to obtain a successful separation in affinity chroma- tography. For instance, it is necessary to immobilize the ligand onto or within a chromatographic support. The immobilization process should ideally create a sta- ble affinity ligand without significantly altering the binding properties of the ligand. If the affinity column is to be reused for multiple cycles, care must also be taken to select application and elution conditions that allow both effective dissociation of retained targets and good column regeneration without permanently damaging the ligand. As an example, much engineer- ing has recently gone into modifying the Protein A li- gand to ensure it can withstand the stringent cleaning and sanitization procedures required for use in a phar- maceutical setting without becoming inactive and no longer being able to bind its target. Similarly, a ligand should not bind its corresponding analyte too strongly, such that the target analyte cannot be eluted without using conditions that damage the analyte. These issues represent areas of ongoing research in the development of affinity ligands. Continued growth is expected in the future for affinity chromatography as further ad- vances are made in the ligands, supports, immobiliza- tion schemes, and potential applications. Recent work has explored the use of molecularly im- printed polymer surfaces (MIPS) for the purification of proteins. MIPS are polymer stationary phases that contain pockets or cavities specific to the target ana- lyte. Due to the lower cost of these surfaces compared with biological affinity ligands, developments in this area are expected to continue and be highly beneficial. PharmTech: What role do you see affinity ligands playing in the future of downstream purification, espe- cially with emerging biologic therapeutic modalities? Bekker (BioCina): Due to their specificity and high purification factor, affinity ligands will continue to play a major role in the downstream purification of biological molecules. Cell affinity chromatography is another important application involving target pu- rification by affinity-based separations. Examples of affinity ligands that have been used for this purpose are lectins, which can interact with glycoproteins on cell membranes, and antibodies, which can bind to specific surface proteins. With the expected increase in cell-based therapies, the development of ligands for these purposes should see them continue to find a large role in the future. Elliott (BioCina): Recently, oligo-deoxythymidine acid (oligo-dT) ligands have been developed and im- mobilized to chromatography stationary phases to pro- vide a highly specific ligand for the polyadenylated tail of mRNA [messenger RNA] molecules. The emergence of this class of therapeutic molecules (mRNAs) drove the development of the corresponding affinity ligand that could simplify their purification. As with cell ther- apies, this new ligand will play an important role in mRNA's future development. Shamlou (Abzena): Gene therapy is an emerging therapeutic modality that needs significant process development including the application of novel affinity ligands during purification. A key step in the produc- tion of gene therapy products is the use of high-qual- ity viral vectors with [AAV] vector and lentiviral vector (LV) being the most popular platforms. Robust manu- facture of high-quality vectors has proven to be a major bottleneck in the growth of gene therapy. Titers are rel- atively low during production and robust purification of the viral particles from impurities has proven to be a challenge. For example, viral particles are physically much larger than proteins and antibodies. Using tradi- tional resins with small pore size leads to low binding capacity because of very low diffusion rates into the pores of the resin. LV particles are also exceptionally sensitive to pH, salt concentration, shear, and tem- perature making their purification more challenging. These challenges currently mean there is no 'platform' downstream purification for viral particles. A new generation of vector serotype specific and non-specific affinity ligand chromatography resins are proving successful in capturing LV and AAV par- ticles from cell-free media and promise the potential of a platform approach to purification of AAV and LV particles. AAV and LV affinity ligands are, however, exceptionally expensive making commercial scale an issue for many drug manufacturers. ■